Antimicrobial peptides have historically been a primary focus of peptide research (Hancock, R. E., and Scott, M. G. 2000. Proc. Natl. Acad. Sci. USA 97: 8856-8861). Their size makes them amenable to detailed structure-activity studies, and the measurement of their cellular efficacy is relatively simple, even if assay conditions have to be adjusted to fit peptide antibiotics (Cudic et al., 2002. Peptides 23: 271-283). Naturally, the most active derivates were considered viable alternatives to small molecules in antimicrobial drug therapy (Zasloff, M. 2002. Nature 415: 389-395).
Resistance induction is rarely seen with peptide-based antimicrobials compared to traditional antibiotics (Ge, et al., 1999. Antimicrob. Agents Chemother. 43: 782-788), but their parenteral use is occasionally hampered by inadequate safety margins and frequently by rapid clearance, leaving them suitable only for topical applications (Bush et al., 2004. Curr. Opin. Microbiol. 7: 466-476). A designer proline-rich peptide dimer was recently described, A3-APO, that kills bacteria by a dual mode of action, and thus is able to kill multidrug resistant clinical isolates of Enterobactericeae in vitro, in concentrations acceptable for clinical development (Otvos et al., 2005. J. Med. Chem. 48: 5349-5359). A3-APO, one of the most potent peptide antibiotics to date, appears to combine the positive features of non-toxic membrane-active antibacterial peptides (Chen et al., 2005. J. Biol. Chem. 280: 12316-12329) and those acting on intracellular targets (Cudic and Otvos, 2002. Curr. Drug Targets 3: 101-106). The bacterial target of A3-APO, similar to many other native proline-rich antimicrobial peptides, is the C-terminal D-E helix of the 70 kDa bacterial heat shock protein DnaK (Otvos et al., 2005. J. Med. Chem. 48: 5349-5359; Kragol et al., 2001. Biochemistry 40: 3016-3026; Bikker et al., 2006. Chem. Biol. Drug Des. 68: 148-153).
Typically, the first steps for progressing from in vitro efficacy measurements to in vivo evaluation of activity and ensuing clinical development are the assessments of peptide stability in vitro and pharmacokinetics in vivo. Serum stability is considered the most important secondary screening assay in drug discovery because it can identify peptides that are unstable in body fluids and thus will fail in the development process (Powell et al., 1993. Pharm. Res. 10: 1268-1273). Pharmacokinetics can identify a 1-hour time period needed for full bacterial killing of the proline-rich peptides with intracellular targets (Cudic et al., 1999. Eur. J. Biochem. 266: 559-565), when the peptide concentration in serum exceeds 130% of the in vitro minimal inhibitory concentration (MIC) value, a minimal dose required for in vivo efficacy (Bush et al., 2004. Curr. Opin. Microbiol. 7: 466-476).
The extent of the decomposition of peptide A3-APO gradually increases from in vitro to ex vivo and in vivo in murine models. This necessarily results in a significantly larger dose of A3-APO required for in vivo efficacy studies than it would be calculated from the in vitro stability data. Therefore, a need exists for a stable peptide that effectively kills bacteria and protects mammals from lethal or sublethal infections in vivo, without exhibiting immunogenic properties.